The co-production of biodiesel and methane gas from grease trap waste (GTW) was evaluated andcompared against theoretical predictions of methane production from sole anaerobic digestion of GTW.The GTW was first processed into two separate phases comprised of fats, oil, and grease (FOG) and highstrength wastewater (GTW wastewater). The GTW wastewater was then anaerobically digested in bio-char packed up-flow column reactors to produce methane gas and a low-strength wastewater effluentwhile the FOG phase was set aside for conversion into biodiesel. Anaerobic digestion efficiencies thatyielded chemical oxygen demand (COD) reductions up to 95% and methane headspace concentrationsbetween 60 and 80% were achieved along with FOG to biodiesel conversion efficiencies of 90%. Methaneproduction yields (m3 per kg COD reduced) achieved theoretical maximums with near total depletion ofthe volatile organic acids. High resolution images of biochar samples confirmed extensive coverage withthick biofilm communities. Microbial analysis revealed broad spectrum populations of anaerobic bacteriathat ferment organic substrates to produce acetate, ethanol, and hydrogen as major end products as wellas archaeal populations that produce methane gas. Energy calculations validated the co-production ofbiodiesel and methane gas from GTW as a competitive option relative to its co-digestion with sewagesludge.

2013 Elsevier Ltd. All rights reserved.1. Introduction

Grease trap waste (GTW) is a complex aqueous mixture of fats,oils, grease (FOG) and water. GTW has a high chemical oxygendemand (COD) (between 138 and 375 g COD L1 [1e3]), a largefraction of lipids, and yields a dewatered FOG possessing roughly4.5e6.5 kW h kg1 [4]. Accordingly, dewatered GTW (i.e. FOG) hasenergy value-added potential for beneficial use in incineration,biodiesel production, or anaerobic co-digestion [4]. The FOGcomponent, however, accounts for only between 0 and 15% byvolume of the GTW, with the remaining wastewater still requiringtreatment due to its high strength (w15e20 g COD L1). As such,processes that produce biodiesel from the FOG component of theGTW must also manage a significant wastewater byproduct. More,the FOG phase has a high free fatty acid content, which requires anadditional acid catalyzed pretreatment step in addition to thestitute, University of Hawaii,USA. Tel.: 1 808 956 7337;

y).

All rights reserved.typical alkaline catalyzed transesterification of triglycerides forbiodiesel production [4]. For these reasons, there have beennumerous publications promoting either the direct anaerobicdigestion of GTW or its co-digestion with sewage sludge [5].Anaerobic digestion is a robust process and is widely applied as aprocess to recover energy from biomass and waste [6]. Despite thereported benefits of direct co-digestion, these and other studieshave also reported a wide assortment of operational challenges,such as the inhibition of acetoclastic and methanogenic bacteria,substrate and product mass transport limitation, sludge flotation,digester foaming, blockages of pipes and pumps, and clogging ofgas collection and handling systems [4]. Of these operational con-cerns the inhibition of methane generation as a result of thedegradation products of the lipids in FOG is one of the most com-mon [7].

These challenges pose significant hurdles to the direct or co-digestion of GTW. As pilot studies have shown the conversion ofthe FOG component of GTW to biodiesel is feasible [8]. An alter-native strategy, then, could be to separate and recover the lipid richFOG portion of GTW for use as a feedstock for biodiesel productionand to anaerobically treat the leftover high strength wastewater toproduce methane gas. Although methane has many benefits,

R.J. Lopez et al. / Renewable Energy 62 (2014) 234e242 235biodiesel is a heavy fuel that can be easily blended with diesel tosupport ship, rail, and truck transportation [9]. Biodiesel is also theonly alternative fuel that can be blended (at low concentration)with petroleum fuels to operate on unmodified combustion en-gines [10]. Although many have promoted the production of bio-diesel from dark fermentation of sugars [11], processed sugar mustbe harvested and purified from crops (i.e. sugar cane) which in-troduces sufficient costs to undermine its utility [12]. The produc-tion of biodiesel from microalgae harvested from solar drivenproduction systems is challenged and a long way from reality[13,14]. Finally, biodiesel from oil seed crops challenges food pro-duction [15]. For these reasons it is worth considering the pro-duction of renewable biodiesel and not methane from the lipid richFOG component of GTW. Lipid rich FOG presents a significant newfeedstock market for biodiesel production; approximately 18.12billion kilograms of GTW are generated annually in the UnitedStates, which contains w1.812 billion kilograms per year of recov-erable fats and oils that could produce w1892.5 million liters ofbiodiesel per year [16].

A major biodiesel producer in Hawaii, Pacific Biodiesel, hasrecently commissioned a commercial scale operation that producesbiodiesel from blended feedstocks including FOG high in free fattyacids (FFAs) with conversion efficiencies as high as 90%. In theirapproach the GTW is initially collected in tanker trucks andtransported to a central processing plant where the FOG compo-nent is separated from the wastewater prior to its conversion tobiodiesel. The separated high strength GTW wastewater is thendischarged directly to the sewer system at a cost based oncomposition (i.e. the discharge fees are applied to the levels ofbiological oxygen demand (BOD5) and total suspended solids (TSS)in addition to the volume of water discharged). To reduce thesecosts, it is proposed to treat the wastewater on-site throughapplication of high rate anaerobic digestion to both reduce thewastewater COD and TSS content prior to its discharge to the sewernetwork while simultaneously producing methane gas. Althoughmany studies to date have focused on either co-digestion of GTWwith sewage sludge or the application of high rate anaerobictechnologies to process FOG-like high lipid industrial wastes [4,17],Fig. 1. Schematic of 3-stage packed bed up-flow column anaerobic digester reactor used in(R1); (C) Fixed film column reactor 2 (R2).there remains a paucity of commentary that addresses the anaer-obic treatment of the high strength GTW wastewater component.

Anaerobic digestion of GTW wastewater faces significant chal-lenge as components in food waste can be difficult to metabolize[18]. In this work we examine the approach of high rate anaerobicdigestion using an integrated 3-phase high rate anaerobic digestionreactor system comprised of an initial continuously stirred hydro-lysis tank reactor followed by two biochar filled column reactorsconnected in series. Corn cob biochar has a very high pore densityand good pore size distribution [19,20], two qualities found tosupport growth of active biofilms that retain methanogenic archaeawhen processing wastewaters at relatively high organic loadingrates (OLRs) and relatively short hydraulic retention times (HRTs)[21]. The aim of this work is to investigate the degree to whichGTW wastewater can be processed readily at HRTs approaching 1day with high COD reductions and methane yields (per unitCOD reduced) that approach the theoretical limit (i.e. 0.35 m3 kgCODred) [22]. The energy content of biodiesel recovered from FOG(assuming a 90% conversion) plus the methane recovered fromanaerobic digestion of GTW wastewater is also compared againstthe total energy content that could be recovered solely fromanaerobic digestion of GTW.

2. Materials and methods

2.1. 3-Stage anaerobic digestion system

All experiments were conducted in a 3-phase anaerobic digestercomprised of an initial 3 L continuously stirred hydrolysis mixingtank (HYD, 2 L working volume) followed by two identical 5 Lcolumn reactors (R1 and R2, each of 4 Lworking volume) connectedin series (Fig. 1). Internal recycle was used to provide intermittentmixing within each column via pumping (Greylor Co.12 V AC pumpmodel PQM-1/115) reactor fluid removed from just below thesurface and pumping it back into the bottom of the reactor. Thesystem HRT was controlled by peristaltic pumping (Watson-Mar-low model 323s) of feed into the HYD tank through silicone tubing(ColeeParmermodelMasterflex 94610-14). Flow rate from the HYDthis study. Symbols: (A) Hydrolysis mixing tank (HYD); (B) Fixed film column reactor 1

R.J. Lopez et al. / Renewable Energy 62 (2014) 234e242236tank to and throughout the column reactors was through gravitydriven hydraulic flow. Both columns were connected to each otherthrough 3/400 PVC piping. The topmost apex of the final exit pipewasmade adjustable in order to permit control of liquid levels in allreactors. The connecting lines were also tapped with venting portsto exhaust gas produced during liquid transfer between reactors.During the inoculation phase on synthetic media a small portion ofthe total system flow (w5%) was recycled from the top port (P3) ofR2 to the bottom port (P1) of R1 or to the HYD tank through use of agear pump (Cole Parmer model 75211-10).

Temperature control in the mixing tank was accomplishedthrough use of a cartridge heater inserted through the top platethrough which voltage was controlled via an external controller (B.Braun Biotech International model Micro DCU Twin). pH in themixing tank was maintained at 37 C and 6.0e6.5, respectively, viaan external controller (B. Braun Biotech International model MicroDCU Twin) using 2 M NaOH as the base addition. The HYD tank saton top of a stir plate which operated continuously to maintainhomogenous mixing. The temperature of both columns was kept at37 C by an external peltier water heater (Varian model Cary PCB150) which pumped heated water through plastic tubing thatpassed around each column reactor. Foam jackets were placedaround both columns to reduce radiative heat loss. A 12 V DC pump(Greylor Co. model pq-12DC) attached with plastic tubing to thebottom of each column was run twice a day for 10 s (displacingapproximately 200 ml) to recycle solids that had settled in thecolumns back to the mixing tank.

2.2. Biofilm supports

The biofilm support material was corn cob biochar produced byflash carbonization (FC) [19]. Intact, carbonized corn cobs werebroken down into pieces possessing an average volume of 2e3 cm3.Trials were run at low and high biochar packing density. The lowpacking density trials utilized three filter discs inserted into both(R1 and R2) of the column reactors, about 10 cm apart. Each disccontained 7.0e7.5 g of immobilization material, loosely packed intoa single layer and held in place between two highly porous sheets ofplastic grating that were secured together with wire. The discs hadnearly the same outside diameter (w10 cm) as that of the inside ofthe reactor columns. In total, each column held approximately 22 gof biochar confined to the filter discs, resulting in a packing densityof 5.5 gbiochar L1 per total reactor volume. The high packing densitytrials incorporated 100 g of biochar (average sizes of 2e3 cm3)packed into a single cylindrical basket of diameter equivalent to theinside diameter of each column reactor and displacing a volumeequaling 1.23 L, yielding a packing density of 75 gbiochar L1 perbasket or 25 g L1 over the entire reactor volume. The baskets weremade of the same highly porous, plastic grating used to make thefilter discs, and were secured into each reactor using wire.

2.3. Feed media

The synthetic feed (Table 1), shown previously to stronglysupport anaerobic digestion [23], was prepared as reported exceptthat the glucose was replaced with equivalent parts of sucrose. Thesynthetic feed was autoclaved at 121 C for 30 min and thenallowed to cool before being added to the system. GTWwastewaterwas recovered from GTW as follows. GTW was collected directlyfrom tanker trucks as they arrived at a local waste trap greaseprocessing facility. Upon collection the GTW was heated to 70 Covernight to promote its stratification into three phases: a top FOGphase, a middle high strength wastewater phase, and a bottomsolids phase. The heating is required to recover all lipid bearingcomponents that exist as solids at the lower temperatures andwould otherwise remain in the water phase. The GTW wastewaterwas then transferred by peristaltic pumping to a second identicalseparation column and allowed to sit for an additional 24 h at 74 C.Thewastewater was separated from any additional bottom solids ortop oil phase and then stored at 4 C until all wastewater waspooled together and homogenized. The accumulated GTW waste-water was then further distributed into 1 L aliquots that werefrozen at 15 C until thawed and added to an external feed tankthat was fed into the first mixing tank as described above. Thisprocess was repeated throughout all experiments and each newbatch of GTW wastewater was characterized independently.Chemical analysis showed that the GTW wastewater (Table 1)possessed a COD:N:P range above the theoretical minimum of100:2.0:0.28 proposed as necessary to support anaerobic digestion[24] and was thus treated without amendments or modification.

2.4. Sampling

Two samples (30 ml each) of the pretreated feed were collectedat various times during the experiment for analysis and validationof consistency. Effluent from the HYD tank was sampled periodi-cally through a single submerged sample tube while the (R1) and(R2) column reactors were sampled from ports protruding from thesides of columns. In all cases 10 ml of effluent was discarded fromeach port prior to taking two 30 ml samples that were transferredinto individual 60 ml falcon tubes. The headspace gasses of all re-actors were sampled individually using a 60 ml syringe to extractthe gas through a septum sealed port located on the top platedirectly above each headspace. The headspace gas samples wereprocessed immediately by direct injection onto a pre-calibrated gaschromatograph (GC).

2.5. Liquid phase measurements

Soluble samples were centrifuged at 6500 rpm for 10 min at15 C (Beckman Coulter model Allegra 25R). The supernatant fromthe centrifuged sample was then decanted and frozen until thawedand analyzed. Total samples were frozen directly until thawed andanalyzed. Both the soluble and total samples were tested for COD,total nitrogen (TN), and total phosphorous (TP) using a sampleincubator (HACH model DRB 200) and colorimeter (HACH modelDR/890). The total samples were also tested for total suspendedsolids (TSS) and pH and the soluble samples were tested for totalvolatile organic acids (TVOA). Samples were processed according tothe HACH Methods 8000 (HR), 10072 (HR), 8190, and 8196 forCOD, TN, TP, and total volatile organic acids (TVOA), respectivelyusing kit numbers 24159-25, 27141-00, 27426-45, and 22447-00,respectively. Accuracy of all testing was confirmed with standards

Carbon dioxide and methane concentrations of the headspacegas were determined using two column GC (Agilent Technologies6890) as previously described by Cooney et al. [23]. Gas productionrates were executed using a 3-channel, solenoid actuated valve gassensor (gas box) that was connected to ports exiting the headspacesof each reactor as described previously [25]. The system wasassumed to have achieved steady state after two residence timesunder constant conditions (i.e. constant OLR, HRT, pH, and tem-perature) and after consistent gas production.

2.7. Scanning electron microscopy

Samples of bare and biofilm coated biochar were viewed byscanning electron microscopy (SEM) using a field emission scan-ning electron microscope (Hitachi S-4800). The biofilm coatedsamples were soaked overnight in 4% glutaraldehyde in 0.1 Mcacodylate buffer (pH 7.4) and then rinsed in progressivelyincreasing concentrations (10, 20, 30, 50, 70, 85, 95, and 100%) ofethanol before being dehydrated in a critical point dryer. Bare andbiofilm coated samples were then fixed on conductive stubs andthen sputter-coated with goldepalladium for 50 s (Hummer 6.2)prior to placement into the SEM chamber after which an acceler-ating voltage of 10 kV was used to take images at various levels ofmagnification.

2.8. Microbial analysis

The bioreactors were drained and the biofilm coated biocharlaid out on absorbent paper. The samples were then cut into piecesof approximately 0.5 cm3 and then stored overnight at 4 C untilextracted for microbial analysis. Total genomic DNA was extractedusing the UltraClean DNA extraction kit (MoBio) according to themanufacturers instructions, and the extracted DNA was storedat 20 C before use. Microbial community structures wereTable 2Characteristics of up flow anaerobic fixed film reactor modules on synthetic and GTWwasat 37 C.

Cloning and sequencing were conducted to obtain sequenceinformation for the major bacterial and archaeal populationsidentified by PCReDGGE. Bacterial and archaeal 16S rRNA geneswere PCR amplified and ligated into pGEM-T Easy cloning vector(Promega) according to the manufacturers instructions. The liga-tions were transformed into Escherichia coli DH10B cells by elec-troporation, and clones with DNA inserts were picked followed bysubsequent screenings. The clones were first screened for insertsusing flanking vector primers, M13F (50-GTTTTCCCAGTCACGAC-30)and M13R (50-CAGGAAACAGCTATGAC-30). Clones containing in-serts were then screened by PCReDGGE alongside the communitysamples to identify clones corresponding to unique bands in thecommunity samples. The clones were sent to the ASGPB DNAsequencing facility at the University of Hawaii at Manoa forsequencing. Sequences were trimmed and quality checked usingSeqMan (DNA star). Homologies of the 16S rRNA gene sequences toknown sequences were retrieved using BlastN in August 2012 [28].

2.9. System start-up

The reactors were first inoculated at low biochar packing den-sity with a total of 8 L of deionized (DI) water, 300 ml of activatedanaerobic sludge obtained from a local wastewater treatment plant,and 1.0 L of synthetic feed media. The system was then grown onthe synthetic feed for 79 days at a constant feed rate of 1.0 L day1,corresponding to an HRT of 10 days and an organic loading rate(OLR) of 1.53 kg COD m3 d1. After the low packing density trials,the transition to high packing density experiments was executed asfollows: The reactor liquid was drained and collected and the discstewater on corn cob biochar as a function of packing density and organic loading rate

R.J. Lopez et al. / Renewable Energy 62 (2014) 234e242238removed. The biochar in these discs was then added, along withadditional new biochar, to containment baskets which were re-inserted into each column reactor. The columns were then refil-led with the drained liquid and the system was fed GTW waste-water for a period of two months during which time the HRT wasreduced incrementally from ten to three days.

3. Results and discussion

3.1. System start up

Within days of the inoculation biomass could be seen colonizingthe surface of the biochar and samples observed under 100magnification confirmed the abundance of rod-shaped bacterialikely containing representatives from genera including; Bacter-oides, Clostridium, and Escherichia contained in the sludge used toinoculate the digester [29]. Over the course of 79 days, the syntheticfeed ratewas increased to 1.45 L d1 resulting in and HRTof 7.3 daysand an OLR of 2.2 kg COD m3 d1 until steady state was verifiedafter approximately two months. At steady state, the reduction inCOD achievedwas 50% (total) and 73% (soluble) in the effluent fromthe HYD reactor, 63% (total) and 79% (soluble) in the effluent fromR1, and by 75% (total) and 85% (soluble) in the effluent of R2, withmethane headspace concentrations of 46.8, 52.8, and 64.2%,respectively above HYD, R1, and R2 reactors. The specific CODremoval rate increased across all reactors from 1.1 kg COD m3 d1

(1.61 soluble) in the HYD reactor to 1.39 kg COD m3 d1 (1.74soluble) in R1 to 1.65 kg COD m3 d1 (1.87 soluble) in R2. TheTVOA rose from 390 mg L1 (in the sucrose rich synthetic feed) to1400 mg L1 in the HYD reactor and to 2080 mg L1 in R1 beforedropping back down to 990mg L1 in the effluent fromR2. Total gasand methane production rates (0.585 and 0.273 m3 m3 d1) werehighest in the HYD reactor. Finally the overall methane yield perkilogram of COD reduced achieved the theoretical limit at0.35m3 kg per kg of CODred. These results confirmed an appropriatepopulation of methanogenic microorganisms in the biofilms ofboth reactors as well as a distribution of phase across the systemwhere acidogenesis was pronounced in the HYD reactor, acido andacetogenesis in R1 and methanogenesis in R2.

The system was then converted to high strength GTW waste-water and the HRT reduced in increments to 3 days, yielding an OLRof 5.3 kg COD m3 d1. This HRT was chosen as the systems upperlimit in order to meet our industry collaborators desire to processthe wastewater in three days (or less), the step down in HRT from7.3 to 3 days was executed as a means to allow the microbialcommunities within the biofilm to adjust to the new feed andhigher OLR. At steady state the performance followed trends similarto operation on synthetic feed although the total COD reductionwas less efficient at 12.4% (9.4% soluble) in the HYD reactor, 35%(53% soluble) in R1, and 45% (59% soluble) in R2. The specific CODreduction increased with each reactor from 1.16 (1.8 soluble) kgCOD m3 d1 in HYD to 1.83 (2.6 soluble) kg COD m3 d1 in R1 to2.37 (2.9 soluble) kg COD m3 d1 in R2. The TVOA increased fromHYD to R1 from 1.93 to 2.98 g L1 as acidogenesis was active beforedeclining by the effluent of R2 to 1.92 g L1. Total gas productionrates decreased with each reactor from 0.63 m3 m3 d1 in HYD to0.43 m3 m3 d1 from R1 to 0.15 m3 m3 d1 from R2. Methane gasproduction rates followed this trend with values of 0.339, 0.297,and 0.11 m3 m3 d1 in HYD, R1, and R2, respectively. Finally, themethane gas production yield on COD reduction was 0.38 m3

produced per kg COD reduced, confirming an appropriate distri-bution of healthy and active populations of aceto, acido, andmethanogens within all reactors although the biochar packingdensity was too low to execute complete digestion on the morecomplex feed at higher OLRs.3.2. Treatment of GTW wastewater

Performance at a packing density of 75 g L1 was explored atHRTs of 3, 2, and 1 day and corresponding OLRs of 5.53, 9.75, and21.2 kg COD m3 d1 (Table 2). The system performed well at allHRTs, achieving total and soluble COD reductions of 81 and 94%,respectively, for an HRT as low as 1 day. Optimal performance interms of COD reduction was achieved at a two day HRT, however,with COD reductions of 92% (total) and 95% (soluble) realized.More, the reduction in TSS dropped to only 13% at an HRT of only 1day, a value significantly below reductions of 75 and 76% achievedat HRTs of 2 and 3 days, respectively. This sharp drop off in TSSreductionwas largely seen as the break point where the packed bedcolumns became unable to retain biofilm entrained bacteria as wellas undigested solids that were otherwise metabolized at the higher

R.J. Lopez et al. / Renewable Energy 62 (2014) 234e242 239HRTs. At all HRTs the reduction of TVOAs present in the GTWwastewater feed was largely accomplished with levels as high as98% being achieved (Fig. 2A). In general the reduction was morerapid and efficient at the highest HRT of 3 days (99%) with the leastdecline in efficiency at an HRT of 1 day (91%). Nonetheless, TVOAconcentrations as low as 80 mg L1 (compared to the feed valueof approximately 4250 mg L1) indicated nearly completemetabolism.

Methane (CH4) production rates from each reactor generallyincreased with decreasing HRT (Fig. 2B). The percentage of gasproduction that was methane in the reactor headspace increasedacross the system for all HRTs, with values as high as 78e81%achieved (Fig. 2C), suggesting active methanogenic microbial cul-tures with no significant loss of active biofilm activity even at theFig. 3. SEM images of native and biofilm covered corn cob biochar. Graphs: (A) Macromagnification; (C) 800 times magnification; (D) 2000 times magnification; (E) 2200 timeslowest HRT (and highest interstitial up-flow velocity). Likewise, themethane yields per amount of COD reduced achieved nearmaximum theoretical levels of 0.34, 0.39, and 0.38 m3 per kg CODreduced at each HRT, respectively.

The digestion process did not significantly reduce TN, presum-ably due to an absence of pathways for significant nitrogen uptakeother than those used for anabolic biomass formation. The TPremained mostly stable with a slight decrease attributed to theaffinity of biochar to absorb phosphate PO43 ions. The pH of thewaste stream slightly increased from a base adjusted value of 6.5 inthe mixing (HYD) reactor to an average value of 6.8 in the second(R2) reactor (data not shown), showing a significant capacity of thesystem to maintain a stable pH despite consuming a feed (sucrose)that produced a significant amount of acids.pore structure at 250 times magnification; (B) Wall of a macro pore at 2500 timesmagnification; (F) 100,000 times magnification.

Fig. 4. DGGE gels showing microbial community profiles for the bacteria (panel A) andarchaea (panel B) in the anaerobic reactors (R1 and R2) based on 16S rRNA genes. DNAsequence information was obtained for the numbered DGGE bands. Lanes i and iii arereplicate samples for reactor 1, and lanes ii and iv are for reactor 2.

SEM images of pure biochar revealed a highly porous surfaceconsisting of macropores of roughly 50 microns diameter wherecorn kernels once sat (Fig. 3A). The surfaces of the macropores arelined with micropores of approximately five microns diameter(Fig. 3B). The overall roughness provides a surface that is conduciveto bacterial colonization and growth of biofilm as visuallyconfirmed from images of biochar samples that showed the surfaceto be densely blanketed with biofilm (Fig. 3CeF).3.4. Microbial community structure

Information on the microbial community composition wasneeded to further the development and optimization of anaerobicdigestion systems [6]. To verify the presence of methanogenic mi-crobial communities within the biofilms (Fig. 3CeF), samples weretaken from both the first and second column reactors. We did notdirectly analyze the microbial community structure of the hydro-lysis tank, as it was continuously impacted by the GTWwastewaterfeed and was not expected to maintain a steady-state microbialcommunity. The two column reactors exhibited identical bacterialcommunity structure as revealed by 16S rRNA gene-based PCR-DGGE analysis (Fig. 4A). Samples obtained at different dates fromTable 3Phylogenetic affiliation of major microbial populations identified by DGGE.

a Percentage identity (ID) of the clone sequence compared to its nearest neighbor.the same reactors (Fig. 4A: lane i versus lane iii, lane ii versus laneiv) also exhibited highly similar microbial structure, indicating thatstable bacterial communities were maintained in the biofilms overtime. The two column reactors also contained several majorarchaeal populations that were shared between the two columns(Fig. 4B). The archaeal community structure also exhibited limitedchange, as indicated by the similar banding patterns over time(Fig. 4B, lane i versus lane iii and lane ii versus lane iv).

DNA sequence information was obtained to determine thephylogenetic affiliation of some major bacterial and archaeal pop-ulations detected by PCR-DGGE (Fig. 4). The nine major bacterialpopulations include six Firmicutes, two Thermotogaceae, and oneSpirochetes (Table 3). All the Firmicutes populations are Clostridia, agroup of anaerobic bacteria that ferment organic substrates to pro-duce acetate, ethanol, and hydrogen asmajor end products [30e32].The two Thermotogae populations are also likely important con-tributors to the anaerobic digestionprocess, asmost cultured speciesof Thermotogae are obligate fermenters of sugar and other complexorganics and produce lactate, acetate, ethanol, and hydrogen asmajor end products [33e38]. As one would expect for anaerobicdigesters, all of the archaeal populations aremethanogens, includingtwo Methanobacteriales and two Methanomicrobia.

3.5. Energy analysis

To evaluate the relative advantage of producing both biodieseland methane gas from GTW, as compared against producing onlymethane gas, the energy produced from each process was esti-mated and compared. Both analyses began with 100 L of GTW thatwas assumed to contain 10% FOG and 90% high strength waste-water possessing a COD of 20 g L1. The assumed conversion effi-ciency of FOG to biodiesel, taken to be 90%, is based upon actualyields achieved (personal communication, Pacific Biodiesel engi-neers), 9 L of biodiesel was produced, yielding 306MJ (assuming anenergy density of 34 MJ per liter). Assuming a COD of 20 g L1, 90 Lof wastewater yields 1.8 kg of COD L1. Using the theoretical yield of0.35 m3 methane gas produced per kg of COD reduced, a valueachieved in this work, 90 L of wastewater produced roughly0.63 m3 of methane gas. Assuming an energy density of 49 MJ m3

for methane gas yields a total energy harvest of 24.57 MJ.Combining this with the energy harvest frombiodiesel yields a totalproduction of 330.57 MJ from 100 L of GTW.

The energy that could be produced solely from anaerobicdigestion of the entire 100 L of GTW was calculated by estimatingthe methane produced if the FOG and GTW wastewater compo-nents were treated separately and then taking their sum. Themethane produced from anaerobic digestion of the GTW waste-water was calculated as above (i.e. 24.5 MJ). The methane producedIdentitya Phylum Domain

R.J. Lopez et al. / Renewable Energy 62 (2014) 234e242 241from anaerobically digesting 10 L of FOG was estimated by usingthe theoretical methane potential value for fat (1014 ml CH4 pergram volatile solids [5]). Assuming that 10 L of FOG produces8478 gVS (assuming a density of 7.5 kg per gallon and 0.942 gVS pergTS [3]) the total methane produced is 8596.6 L of methane whichyields 335.2 MJ. When added to the production from the GTWwastewater the total methane production becomes 359.7 MJ, avalue that is slightly higher than the 330.57 MJ produced from co-production of biodiesel and methane. However, it is not likely thatthe theoretical methane potential for pure fat will be achieved as itis known that the FOG contains roughly 10% of materials that areinsoluble or unsaponifiable. In this case a more reasonable estimateof the methane production is 845 ml of methane produce per gramof volatile solids consumed [7]. In this case only 7164 L of CH4 areproduced which, using the yield of 0.942 gVS per gTS [3] yields atotal energy production of (279 24.5) 303.5 MJ e a value that islower than when processing biodiesel and methane gas from GTW.

4. Conclusions

GTW wastewater was successfully treated at HRTs as low as 1day and OLRs as high as 21.2 kg COD m3 d1 using biochar filledpacked bed column reactors connected in series and fronted by aninitial hydrolysis reactor that buffered the system fromdisturbancesin pH and temperature. Reductions in COD as high as 95% wereroutinely achieved as were methane headspace gas compositionsabove 70% (and some above 80%). Methane production reachingmaximum theoretical yields of 0.35m3 per kg CODredwere achievedalong with reductions in TVOAs above 98% as a result of fatty acidproduction via the EmbdeneMeyerhofeParnas (glycolysis)pathway (i.e. the oxidation of sucrose to pyruvate), the reduction ofpyruvate to butyrate or propionate and/or the oxidation of pyruvateto acetate [39], and the production of acetate directly from CO2 andH2 via the WoodeLjungdahl (reductive acetyl-CoA) pathway[40,41]. The TSS reduction however, dropped to below 15% at thehighest HRT of 1 day, suggesting that the digester had reached abreak point capacity despite the soluble COD still being almostentirely digested. Microbial analysis revealed broad spectrumpopulations of anaerobic bacteria that ferment organic substrates toproduce acetate, ethanol, and hydrogen as major end products aswell as archaeal populations that produce methane gas.

The process of co-producing biodiesel and methane gas fromGTW was shown to produce as much or more energy than theprocess of producing methane gas solely from anaerobic digestionof GTW. Combining this result with the realization that biodieselcan be blended with diesel, and that renewable sources of biodieselfrom oil seed crops or sugars via dark fermentation is environ-mentally and economically challenged, suggests that treatmentstrategies for GTW that produce both biodiesel and methane gasshould be strongly considered relative to those that produce onlymethane gas.

Acknowledgments

Funding this work was provided by the DOE (#DE-EE0003507,R. Rocheleau, PI) and a University of Hawaii sustainability grant outof the Office of Vice Chancellor for Research and Graduate Educa-tion. The authors also thank Pacific Biodiesel for providing the GTWand Bob King for his input.